A lateral flow assay (LFA) system is mainly used as a method for rapidly measuring an antigen-antibody reaction. It is general in the LFA that an antibody is fixed to a membrane in which a liquid sample enables to flow due to a capillary phenomenon, wherein the membrane has a conjugate pad and a sample pad connected on an upper portion thereof and an absorption pad connected on a lower portion thereof. The conjugation pad contains a dried gold nanoparticle conjugate to which an antibody capable of selectively binding to a sample material is fixed. In the membrane, materials capable of binding an antibody selectively reacting with the sample material and capable of binding the antibody fixed to the gold nanoparticle are fixed at different positions, respectively. The antibody fixed to the membrane and the antibody fixed to the gold nanoparticle, which are capable of selectively binding the sample material, are configured so as to bind to each other in a sandwich shape with respect to the sample material. The absorption pad consists of a material capable of favorably absorbing the liquid sample. In the case where a liquid sample solution drops onto a sample pad in the above-mentioned LFA system, when a sample is present, antibodies having selectivity on the sample, that is, antibodies fixed to the gold nanoparticle and the membrane are bound to each other in a sandwich shape, thereby forming a band capable of confirming with the naked eye at a position of the membrane having the antibodies fixed thereto.
As a membrane sensor, there are various published documents such as Korean Patent No. 599420 entitled with Membrane Strip Biosensor System for Point-of-CareTesting; Japanese Patent Laid-Open Publication No. 2006-507511 entitled with Composite Sensor Membrane; Korean Patent No. 348351 entitled with Electrochemical Membrane Strip Biosensor; U.S. Pat. No. 7,494,818 entitled with Method for Determining Concentration of Multiple Analytes in a Single Fluid Sample; Korean Patent No. 591390 entitled with Sensor Having Membrane and Method For Manufacturing the Same; US Patent Application Publication No. 2005-214161 entitled with Test Device for Simultaneous Measurement of Multiple Analytes in a Single Sample, and the like.
However, the existing LFA system using nanoparticles requires improving a detection sensitivity due to a low detection sensitivity of 1 ng/ml and has a disadvantage in that it is difficult to implement a high sensitivity reaction such as enzymatic color reaction, fluorescence, chemiluminescence, and the like, except for a sandwich method using nanoparticles, by one-step sample injection. In addition, in the case of a bio reaction required for two or more stages of reaction conditions such as an enzyme reaction and a chemiluminescent reaction, an LFA strip has a difficulty in obtaining a desired result by a single sample injection, and in a case of a chemiluminescent strip, 2 step method in which a luminol reaction solution is secondarily injected for a luminol reaction after an antigen-antibody reaction is primarily achieved, or a technology of positioning a support having a luminol reactant applied onto a reaction membrane and being contact the support with a reactant part at a time of completion of an antigen-antibody reaction (EP No. 1,9652,212 and US Patent Application No. 2009-142781) have been known so far.
Therefore, development of a strip capable of measuring a bio reaction having two or more stages of reaction conditions by one-step process through a single sample injection has been demanded.